The endothelium is an important component of normal vascular homeostasis, in part, through its production of nitric oxide (NO?) via the endothelial isoform of nitric oxide synthase (eNOS). In patients with cardiovascular disease, impaired endothelial NO? bioactivity leads to a loss of the normal endothelial inhibition of platelet activity and inflammation, predisposing patients to clinical cardiovascular events such as heart attack and stroke. This impairment of endothelial NO? bioactivity is thought secondary to increased vascular levels of reactive oxygen species (ROS) that chemically destroy NO? and promote a maladaptive vascular phenotype. Vascular ROS are produced by a number of enzymes, although the NADPH oxidase (Nox) family appears important for endothelial dysfunction. However, recent data indicate that some Nox isoforms are active in "normal" vasculature and Nox4 is particularly abundant in the endothelium. In this application, data are presented that Nox4 promotes endothelial NO? bioactivity and is important for normal phenotypic responses such as endothelial proliferation and angiogenesis. Thus, the central hypothesis of this proposal is that Nox4 promotes normal endothelial function. To address this hypothesis, the role of Nox4 in modulating eNOS will be characterized and underlying mechanisms probed including Akt, AMP kinase, and protein tyrosine phosphatase 1B. Subsequently, the role of Nox4 on modulating phenotypic endothelial responses important for angiogenesis such as proliferation, migration, and recovery from hindlimb ischemia will be examined. The work contained in this proposal will be important for defining the molecular events that distinguish adaptive ROS-mediated responses from those that are maladaptive and contribute to cardiovascular disease. As a consequence, this work will help provide the necessary insight to design new treatments for cardiovascular disease, the principal source of morbidity and mortality in the developed world.